Project Summary Neuromyelitis optica spectrum disorder (NMOSD) is a severe autoimmune disorder targeted against the aquaporin-4 (AQP4) water channel. Central nervous system (CNS) injury in NMOSD is initiated by the binding of AQP4 autoantibodies (AQP4-IgG) to target astrocytes and the activation of antibody effector functions; however, serum AQP4-IgG titers do not correlate with disease relapse or severity. Therefore, identifying factors that influence CNS access, intrathecal production, or effector function of AQP4-IgG is essential for understanding NMOSD pathognesis. Using single-cell sorting, recombinant antibody technology, and heavy-chain variable region repertoire analysis, we have reconstructed the intrathecal AQP4-IgG response in NMOSD, isolated anti-endothelial antibodies that activate brain microvascular endothelial cells (BMECs), and identified an expanded CD27-IgD- double negative (DN) memory B cell population clonally related to intrathecal AQP4-specific plasmablasts. We are now uniquely prepared to test our hypothesis that AQP4-IgG, autoantibodies against BMECs, and pro-inflammatory DN B cells act in concert to propel NMOSD activity and pathology. In Aim 1, we will examine the contribution of glucose regulated protein-78 (GRP78) autoantibodies to NMOSD disease activity. We will investigate the abundance and epitope specificity of NMOSD anti-GRP78 autoantibodies, evaluate the cell signaling pathways activated in BMECs, and measure their ability to increase vascular permeability in the brain and retina. In Aim 2, we will address how the binding specificity of individual anti-AQP4 autoantibodies affect lesion formation and CNS injury in NMOSD. We have identified multiple species of patient-derived AQP4-specific recombinant antibodies that produce distinct effects on target astrocytes. We will gauge AQP4 rAbs on their ability to activate complement dependent cytotoxicity, initiate antibody-dependent cell-mediated cytotoxicity, and modulate astrocyte chemokine and cytokine production in vitro. We will then evaluate how these parameters affect NMOSD lesion formation in animal models and disease activity in affected patients. And in Aim 3, we will analyze an expanded population of class-switched CD27-IgD- DN B cells in NMOSD patients and investigate their role in disease activity. The abundance and immunoglobulin repertoire of DN B cells will be compared during NMOSD relapse and remission, and we will assess the cytokine response and immunoglobulin production of NMOSD DN B cells in response to various stimuli. In addition, we will investigate the potential for NMOSD DN B cells to present AQP4 to autologous T cells. The results of these investigations will elucidate how AQP4-IgG accesses the CNS, how intrathecal B cells contribute to NMO lesion initiation, and how discrete subpopulations of AQP4-IgG contribute to CNS injury. The outcomes of our research will remove barriers to progress in the field and advance approaches to the diagnosis and treatment of NMOSD and other autoimmune disorders impacting the CNS and eye.